ABSTRACTThe eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

Figure 5: MARCH-1 and -8 interact with and down-regulate wild-type TRAIL-R1, but not the TRAIL-R1 K273A mutant.A, MCF-7Casp-3 cells were transfected to express mRFP only (−), mRFP-tagged WT TRAIL-R1 or K273A (K/A) mutant, together with HA-tagged MARCH-1, MARCH-8, or empty vector (−), as indicated. Immunoprecipitation was performed with α-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting with α-mRFP and α-HA antibodies to detect TRAIL-R1 and MARCH-1/8, respectively. Panel I, mRFP detection in IP of TRAIL-R1.mRFP and control mRFP; panel II, MARCH-1 and -8 detection in IP of TRAIL-R1.mRFP and control mRFP; panel III, mRFP detection (TRAIL-R1.mRFP or RFP only) in total cell lysates (TCL); panel IV, MARCH-1 and -8 detection in TCL. B, quantification of TRAIL-R1 down-regulation in total cell lysates. Total protein levels of WT TRAIL-R1 and the K/A mutant in TCL of control cells (−), or those expressing HA-tagged MARCH-1 or -8 were quantified from Western blots as depicted in panel III of A and plotted as percentage of the WT TRAIL-R1.mRFP expression in control cells. Data represent mean ± S.D. of values from the experiment depicted in A and 2 additional experiments. C, impact of MARCH-1 or MARCH-8 on WT and K/A mutant TRAIL-R1 cell surface expression. Cells stably expressing WT or K273A TRAIL-R1.mRFP were transfected to express GFP (−), GFP-tagged MARCH-1 or MARCH-8, and stained with antibody to TRAIL-R1 as outlined for Fig. 2. Quantification of 2–4 independent experiments assessing TRAIL-R1 MFI in GFP+ cells as the percentage of TRAIL-R1 MFI in GFP− cells, whereby the values in control cells were set at 100%. Values represent mean ± S.D. Asterisks indicate statistically significant differences between TRAIL-R1 WT or K/A mutant (Student's t test; *, p < 0.05, **, p < 0.01).

Mentions:
MARCH-8 is known to interact with its ubiquitination substrate CD86 (16). We therefore examined whether TRAIL-R1 could interact with MARCH-1 or -8. WT TRAIL-R1.mRFP or the K273A mutant were co-expressed with HA-tagged MARCH-1 or -8. Next, the TRAIL receptors were isolated by immunoprecipitation and isolates were immunoblotted with anti-HA antibody to detect associated MARCH proteins. Both MARCH proteins were detected at high stochiometry in the WT TRAIL-R1 immunoprecipitates (IP, Fig. 5A, panels I and II), indicating that TRAIL-R1 forms a complex with MARCH-1 or -8 at steady state. Strikingly, MARCH-1 and -8 interacted to a much lesser extent with the TRAIL-R1 K273A mutant, indicating that Lys-273 enables or greatly strengthens the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 (Fig. 5A, panels I and II).

Figure 5: MARCH-1 and -8 interact with and down-regulate wild-type TRAIL-R1, but not the TRAIL-R1 K273A mutant.A, MCF-7Casp-3 cells were transfected to express mRFP only (−), mRFP-tagged WT TRAIL-R1 or K273A (K/A) mutant, together with HA-tagged MARCH-1, MARCH-8, or empty vector (−), as indicated. Immunoprecipitation was performed with α-mRFP antibody and immunoprecipitates (IP) were analyzed by immunoblotting with α-mRFP and α-HA antibodies to detect TRAIL-R1 and MARCH-1/8, respectively. Panel I, mRFP detection in IP of TRAIL-R1.mRFP and control mRFP; panel II, MARCH-1 and -8 detection in IP of TRAIL-R1.mRFP and control mRFP; panel III, mRFP detection (TRAIL-R1.mRFP or RFP only) in total cell lysates (TCL); panel IV, MARCH-1 and -8 detection in TCL. B, quantification of TRAIL-R1 down-regulation in total cell lysates. Total protein levels of WT TRAIL-R1 and the K/A mutant in TCL of control cells (−), or those expressing HA-tagged MARCH-1 or -8 were quantified from Western blots as depicted in panel III of A and plotted as percentage of the WT TRAIL-R1.mRFP expression in control cells. Data represent mean ± S.D. of values from the experiment depicted in A and 2 additional experiments. C, impact of MARCH-1 or MARCH-8 on WT and K/A mutant TRAIL-R1 cell surface expression. Cells stably expressing WT or K273A TRAIL-R1.mRFP were transfected to express GFP (−), GFP-tagged MARCH-1 or MARCH-8, and stained with antibody to TRAIL-R1 as outlined for Fig. 2. Quantification of 2–4 independent experiments assessing TRAIL-R1 MFI in GFP+ cells as the percentage of TRAIL-R1 MFI in GFP− cells, whereby the values in control cells were set at 100%. Values represent mean ± S.D. Asterisks indicate statistically significant differences between TRAIL-R1 WT or K/A mutant (Student's t test; *, p < 0.05, **, p < 0.01).

Mentions:
MARCH-8 is known to interact with its ubiquitination substrate CD86 (16). We therefore examined whether TRAIL-R1 could interact with MARCH-1 or -8. WT TRAIL-R1.mRFP or the K273A mutant were co-expressed with HA-tagged MARCH-1 or -8. Next, the TRAIL receptors were isolated by immunoprecipitation and isolates were immunoblotted with anti-HA antibody to detect associated MARCH proteins. Both MARCH proteins were detected at high stochiometry in the WT TRAIL-R1 immunoprecipitates (IP, Fig. 5A, panels I and II), indicating that TRAIL-R1 forms a complex with MARCH-1 or -8 at steady state. Strikingly, MARCH-1 and -8 interacted to a much lesser extent with the TRAIL-R1 K273A mutant, indicating that Lys-273 enables or greatly strengthens the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 (Fig. 5A, panels I and II).

Bottom Line:
The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored.Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression.These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.

ABSTRACTThe eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.